The intent of this proposal is to analyze the mechanism by which the papillomavirus E5 oncoprotein transforms cells. E5 is structurally unique among the transforming proteins and the best-studied E5 protein is that encoded by bovine papillomavirus type 1 (BPV- 1). The E5 polypeptide consists of 44 amino acids (the first thirty are hydrophobic) and localizes predominantly to the Golgi apparatus. Recent data indicate that E5 may intercede in the signal transduction pathway since part of the E5 transformed phenotype consists of the activation of cellular growth factor receptors such as EGF, CSF- 1, and PDGF. To date, however, then is no evidence that this is a direct consequence of E5 activity and attempts to isolate E5-associated cellular proteins or identify cellular targets for E5 have been unsuccessful. Using a newly-described methodology of epitope addition, we present preliminary information in this proposal which identifies an E5-associated cellular protein. This protein (designated 16k) specifically associates with E5 during immunoprecipitation and, more importantly, fails to associate with a transformation-defective E5 mutant protein. We propose to utilize E5-epitope fusion proteins to investigate E5/16k complexes at the molecular, biochemical, and biological level. The domains of E5 which participate in 16k binding and Golgi "targetting" will be identified by deletion and site-specific mutagenic analysis as well as the use of specific E5 fusion proteins. E5/16k complexes will be purified, evaluated for their native size and composition, their kinetics of formation and turnover, and used to generate polyclonal and monoclonal antibodies against 16k. Specific antibodies will be used to directly evaluate total cellular 16k for its intracellular localization, synthesis and processing, and potential association with other cellular proteins. In addition, 16k will be affinity-purified and microsequenced. 16k cDNA's will be generated, isolated (using antibodies and oligonucleotides for screening), and sequenced. Wt and mutant 16k cDNA's will be expressed in a variety of host cells to evaluate biological activity. The above in-vitro studies will be supplemented by immunofluorescence studies of frozen tissue to evaluate the in-vivo expression of both E5 and 16k. Finally, similar approaches will be utilized to investigate the related hydrophobic E5 protein of the human papillomavirus, HPV-16. These studies will be important for delineating new control mechanisms of mitogenesis and for dissecting the role of E5 in neoplasia, including human anogenital malignancies in which papillomaviruses play a critical role.